Cross talk between oncogene and ant-oncogene via the Crk oncogene product

Crk癌基因产物激活RAS和RAP1。RAS是一个众所周知的癌基因，而RAP1拮抗RAS依赖的肿瘤发生。为了了解Crk同时激活癌基因和抑癌基因的机制，我们开发了一种基于FRET的单分子探针，可以监测活细胞中Crk的激活。使用这个探针，我们观察到了EGF刺激的细胞中Crk的快速激活。然而，对于原型探针，由于探针的快速扩散，我们无法确定Crk在细胞中的激活从哪里开始。为了克服这一缺陷，我们将Ki-Ras蛋白的CAAX盒融合到探针上，期望探针能够锚定在质膜上。事实上，通过使用这种CAAX融合的探针，我们发现Crk是从细胞的外围区域激活的。此外，我们还构建了表达该探针的重组腺病毒，使我们能够在多种细胞中快速高效地表达该探针。因此，研究Crk活性的新探针的研制，使活细胞中Crk癌基因产物的时空调控可视化，并为我们理解Crk癌基因产物在细胞生长和转化调控中的作用提供了可能。

Crk oncogene product activates Ras and Rap1. Ras is a well-known oncogene, whereas Rap1 antagonize Ras-dependent oncogenesis. To understand the mechanism by which Crk activates both oncogene and anti-oncogene, we developed a FRET-based single-molecule probe that can monitor the activation of Crk in a living cell. Using this probe, we visualized rapid Crk activation in an EGF-stimulated cell. However, with the prototype probe, we could not determine where in the cell the activation of Crk starts, due to rapid diffusion of the probe. To overcome this flaw, we fused CAAX box of Ki-Ras protein to the probe, expecting the anchoring of probe to the plasma membrane. Indeed, by the use of this CAAX-fused probe, we found that Crk was activated from the peripheral region of the cell. Furthermore, we generated a recombinant adenovirus for the expression of the probe, which enabled us rapid and efficient expression of the probe in a variety of cells. Thus, the development of new probe for the Crk activity visualized the spatio temporal regulation of Crk oncogene products in living cells and promised us for the understanding of the role of Crk oncogene products in the regulation of cell growth and transformation.